The present invention is generally directed to detecting phospholipids, and more particularly to using lactadherin or a fragment thereof as an agent for detecting the presence of a phospholipid, such as phosphatidylserine (PS), on a cell membrane.
Lactadherin is a MW 47,000 glycoprotein of milk fat globules. It has also been known as PAS-6/7, indicating the two glycosylation variants (Reference 1), bovine-associated mucoprotein, BA-46, P47, and MFG-E8 (Reference 2). Lactadherin has a domain structure of EGF1-EGF2-C1-C2 in which EGF indicates epidermal growth factor homology domains, and the C domains share homology with the discoidin family including the lipid-binding “C” domains of blood coagulation factor VIII and factor V (Reference 2). The second EGF domain displays an Arg-Gly-Asp motif (Reference 3) which binds to the αvβ5 and αvβ3 integrins (References 1 and 4-6). The second C domain binds to phospholipids (Reference 6).
In milk fat globules, lactadherin lines the surface of the phospholipid bilayer which surrounds the central triglyceride droplet, apparently stabilizing the bilayer (Reference 7). In tissue sections lactadherin is found within the milk ductules and localized on the apical portion of secretory epithelium in the breast (Reference 7). Abundant expression by breast carcinoma makes lactadherin a potential target for antigen-guided radiation therapy (Reference 8). Lactadherin also lines the apical surface of epithelia in the biliary tree, the pancreas, and sweat glands (Reference 7) and is synthesized by aortic medial smooth muscle cells (Reference 9). Lactadherin has been identified as a zona pellucida-binding protein on the acrosomal cap of sperm (Reference 10). Function in these tissues remains unknown. Stimulated macrophages, but not quiescent macrophages, synthesize and secrete lactadherin in vitro (Reference 11). Lactadherin then binds to apoptotic cells expressing phosphatidylserine and mediates phagocytosis of the dying cells via interaction of the lactadherin EGF domain with macrophage integrin(s).
We have recently found that, in vitro, lactadherin functions as a potent anticoagulant (Reference 12). Homology between the lactadherin and factor VIII C domains correlates with efficient competition for membrane binding sites recognized by both factor VIII and factor V. Lactadherin inhibits the factor Xase complex, in which factor VIII functions, and the prothrombinase complex, in which factor V functions. Lactadherin also inhibited the factor VIIa-tissue factor complex and competed with vitamin K dependent factor IXa for membrane binding sites indicating a capacity to block binding sites for non-homologous coagulation proteins. The inhibitory properties of lactadherin contrasted with those of annexin V. Although annexin V binds to phosphatidylserine-containing membranes with high affinity, it functions well as an anticoagulant only when the phosphatidylserine content exceeds 4% and the membrane curvature is very limited. By contrast, lactadherin was an efficient anticoagulant on membranes with <4% phosphatidylserine and regardless of curvature. The in vitro anticoagulant properties of lactadherin suggest the possibility that it may have an anticoagulant function during some physiologic or pathologic conditions (Reference 12).
Blood coagulation factor VIII and factor V bind to phospholipid membranes via “C” domains which share homology with lactadherin “C” domains (References 13-15). Remarkable features of membrane binding include high affinity (KD approx. 2 nM) (Reference 16) and sufficient specificity so that no plasma proteins compete for membrane binding sites (Reference 17). Factor VIII binds via stereo-selective interaction with the phospho-L-serine motif of phosphatidylserine (Ptd-L-Ser) (Reference 18). Factor V also exhibits stereoselective interaction with Ptd-L-Ser (Reference 19). Both proteins associate rapidly with membrane binding sites but have increased affinity conferred by a second, slower binding interaction (Reference 20). Binding of factor VIII is enhanced by the presence of phosphatidylethanolamine (PE) in the membrane (Reference 21), by unsaturated phospholipid acyl chains (Reference 22), and by membrane curvature (Reference 21). The crystal structures of the C2 domains of factors VIII and V suggest that membrane binding is mediated by two pairs of hydrophobic residues displayed at the tips of β-hairpin turns (References 23 and 24). Mutagenesis studies have confirmed the role of these residues in phospholipid binding (References 25 and 26). The homology of the lactadherin C domains with those of factors VIII and V suggests that similar phospholipid binding properties may be mediated by hydrophobic residues on putative β-hairpin turns. Indeed, lactadherin has been found to bind Ptd-L-Ser adsorbed to plastic (Reference 27) and to utilize primarily the C2 domain in its lipid binding (Reference 6). Furthermore, the capacity of lactadherin to quantitatively compete with both factor VIII and factor V for membrane binding sites (Reference 12) suggests that it has high affinity for these membranes and a similar mechanism of binding.
The discovery that lactadherin functions as a anticoagulant was the subject of U.S. Provisional Application Ser. No. 60/386,562, filed Jun. 7, 2002, and International Application No. PCT/US03/15404, filed Jun. 6, 2003, both incorporated herein in their entirety by reference.